Introduction to Vectors

1
Lecture 2 Vectors, cDNA Libraries, Plasmid
Minipreps
2
Introduction to Vectors

• In order to study a DNA fragment (e.g., a gene),
  it needs to be amplified and eventually purified.
• These tasks are accomplished by cloning the DNA
  into a vector.
• A vector is generally a small, circular DNA
  molecule that replicates inside a bacterium such
  as Escherichia coli (can be a virus).

p. 2-1
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Cloning Scheme
Digest
Ligate
Amplify and Prep
p. 2-1
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Vector Types

• There are three commonly used types of vectors
• 1) plasmid vectors (e.g., pUC plasmids)
• 2) bacteriophage vectors (e.g., phage ?) and
• 3) phagemid vectors (e.g., pBlueScriptTM).
• Each has a different use, and there are many
  derivatives of these basic building blocks. In
  this course, you will be using plasmid vectors.

p. 2-1
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Plasmids

• Circular DNA molecules found in bacteria
• Replicated by the hosts machinery independently
  of the genome. This is accomplished by a sequence
  on the plasmid called ori, for origin of
  replication.
• Some plasmids are present in E. coli at 200-500
  copies/cell

p. 2-1
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Plasmid Engineering

• Plasmids also contain selectable markers.
• Genes encoding proteins which provide a selection
  for rapidly and easily finding bacteria
  containing the plasmid.
• Provide resistance to an antibiotic (ampicillin,
  kanamycin, tetracycline, chloramphenicol, etc.).
• Thus, bacteria will grow on medium containing
  these antibiotics only if the bacteria contain a
  plasmid with the appropriate selectable marker.

p. 2-2
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Transforming plasmids into bacteria
p. 2-2
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Safety Features

• Modern cloning plasmids have been engineered so
  that they are incapable of transfer between
  bacterial cells
• Provide a level of biological containment.
• Naturally occurring plasmids with their
  associated drug resistance genes are responsible
  for the recent rise in antibiotic-resistant
  bacteria plaguing modern medicine.

p. 2-3
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Screening for Inserts
p. 2-3
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Plasmid cloning vector pDONR222
p. 2-4
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Cloning a DNA fragment into pDONR222
Transform
Transform
Viable
Lethal
p. 2-4
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DNA Libraries

• DNA library - a random collection of DNA
  fragments from an organism cloned into a vector
• Ideally contains at least one copy of every DNA
  sequence.
• Easily maintained in the laboratory
• Can be manipulated in various ways to facilitate
  the isolation of a DNA fragment of interest to a
  scientist.
• Numerous types of libraries exist for various
  organisms - Genomic and cDNA.

p. 2-5
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Construction and analysis of a genomic DNA
library
p. 2-5
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Construction of a cDNA library
p. 2-6
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Differences between a genomic and cDNA library
Genomic Library Promoters Introns Intergenic Non-e
xpressed genes
cDNA Library Expressed genes Transcription start
sites Open reading frames (ORFs) Splice points
p. 2-7
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Purification of mRNA
Collect and grind up animals in mild denaturing
solution
Spin out debris (Tissue, membranes, etc)
Treat with DNAse (removes DNA)
Treat with Phenol (removes protein)
p. 2-8
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Synthesis of cDNA from mRNA
p. 2-8
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Cloning a DNA fragment into pDONR222
Transform
Transform
Viable
Lethal
p. 2-4
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Preparing Plasmid DNA

• In order to use a vector for cloning, sequencing,
  etc., it is necessary to isolate the vector in a
  highly purified form.
• Routinely done by most labs.
• Many companies now sell kits which provide all
  the solutions necessary for preparing DNA.
• Based on similar procedures

p. 2-10
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Essential components of minipreps

• Gentle lysis step to break open the cells and
  release the plasmid DNA into solution.
• Cell debris and chromosomal DNA of the bacteria
  is pelleted during the centrifugation.
• Plasmid DNA remains behind in the clear
  nonpelleted fraction (the nonpelleted solution
  left after centrifugation is known as the
  supernatant).
• Subsequent steps are then performed on the
  supernatant to remove contaminating RNA and
  proteins from the plasmid DNA.

p. 2-10
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1. Grow the bacteria

• Grow an overnight (ON) culture of the desired
  bacteria in 2 ml of LB medium containing the
  appropriate antibiotic for plasmid selection.
  Incubate the cultures at 37C with vigorous
  shaking.

p. 2-11
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Naming your clones
Year
Your initials
Day
T20AV12.09
Group
Clone
T (Teusday), W (Wednesday), H (Thursday)
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2a. Transfer the cells to a tube and centrifuge

• Transfer 1.5 ml of the culture to a microfuge
  tube and pellet the cells for 1 minute at full
  speed (12,000 rpm) in the microcentrifuge.
• First tap or gently vortex the glass culture
  tube to resuspend the cells which have settled.
  The culture can be transferred to the microfuge
  tube by pouring.

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2b. Remove the supernatant

• Remove the growth medium (supernatant or sup) by
  aspiration or by using the P-1000.
• Leave the bacterial pellet as dry as possible so
  that additional solutions are not diluted.

p. 2-11 (fig not shown)
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3. Resuspend the cell pellet

• Resuspend the bacterial pellet in 250 µl of
  Buffer PI by vigorous vortexing.
• Add 150 ml of PI, cap the tube, and vortex on
  the highest setting (pipetman can be used). Look
  very closely for any undispersed pellet before
  proceeding to the next step. It is essential that
  the pellet be completely dispersed.
• PI contains two essential components Tris and
  EDTA.
• Tris is used to buffer the pH of the cell
  suspension.
• EDTA is a chemical that chelates divalent
  cations (ions with charges of 2) in the
  suspension, such as Mg. This helps break down
  the cell membrane and inactivate intracellular
  enzymes.

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4. Add Solution P2

• Add 250 µl of Solution P2 (0.2 N NaOH, 1SDS),
  mix gently 4-6 times. Do not vortex!! This will
  shear the DNA and contaminate your DNA preps.
• Denatures protein, DNA, RNA, membranes. During
  this step a viscous bacterial lysate forms (the
  cells lyse).

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5. Add Solution N3

• Add 350 µl of Solution N3 (3 M KOAc, pH 4.8).
  Mix gently 4-6 times. Do not vortex.
• P3 neutralizes cell suspension. A white
  precipitate consisting of aggregated chromosomal
  DNA, RNA and cell debris and SDS will form.
• Plasmids will renature

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6. Centrifuge cell debris

• Centrifuge for 10 minutes at full speed in the
  microcentrifuge.
• A white pellet will form on the bottom and side
  of the tube after centrifugation.

p. 2-13
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7. Transfer sup. (DNA) to spin column.

• Using a P-1000 set at 600ul, transfer the
  supernatant to the appropriately labeled spin
  column which has been inserted into the 2 ml
  microcentrifuge tube.

p. 2-13 (Fig not shown)
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8. Centrifuge the spin column

• Centrifuge for 1 minute at full speed, and drain
  the flow-through from the collection tube.

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9. Wash the column with PE

• Add 750 ul of Wash buffer PE to the spin column
  contained in the 2 ml Collection Tube, centrifuge
  at full speed for 1 minute, and drain the
  flowthrough.
• This buffer helps to further remove any nucleases
  that may have co-purified with the DNA. Remove
  the liquid that has passed through the column in
  the same way as performed in Step 9.

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10. Spin the column to remove PE

• Place the spin column in a fresh 1.7 ml
  microcentrifuge tube (with lid cut off) and
  centrifuge again for 1 minute at full speed to
  remove any residual wash solution that might
  still be in the column.
• Any residual wash solution must be removed
  because the ethanol contained in this solution
  may interfere with further DNA manipulations. It
  is normal to remove a small amount of liquid from
  the column at this step, however if a significant
  amount of solution (50-100 ul or greater) is
  found in the collection tube, repeat this step.

p. 2-14 (Figure not shown)
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11. Elute the DNA with EB

• Place the spin column into an appropriately
  labeled 1.7 ml microcentrifuge tube and add 50 ul
  of EB buffer to the column. Centrifuge at full
  speed for 1 minute.
• Elutes the plasmid DNA from the column and
  collects in the microcentrifuge tube.

p. 2-14
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12. Store your DNA

• Remove the spin column from the labeled 1.7 ml
  microcentrifuge tube and close the lid on the
  tube tightly.
• Store the miniprep DNA in your freezer box
  (-20C).

p. 2-14
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Other types of vectors
1. Phage Vectors
p. 2-15
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2. Phagemids
Have plasmid and phage components
F1 Phage origin of replication for making single
strand DNA
p. 2-15
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3. Cosmids - Plasmids with phage packaging sites
p. 2-16
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4. YACs - Yeast Artificial Chromosomes
p. 2-17
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Vector Insert Size
Vector Type Cloned DNA
(Kb) Plasmid 20 Phage 25 Cosmid 45 BAC 300 YAC 100
0
p. 2-17